Co-expression of inhibitory receptors on CD8⁺ and CD4⁺ T cells in chronically HIV-infected BLT mice.

<p><b>A</b>) Representative flow cytometry data of peripheral blood from an HIV-infected BLT mouse at 13 weeks post infection. Co-expression of CD244, CD160, and LAG-3 with PD-1 was determined on human CD8⁺ and CD4⁺ cells. <b>B</b>) Percentages of PD-1 expressing CD8⁺ and CD4⁺ T cells co-expressing CD244, CD160, and LAG-3 at 13 weeks and 26 weeks post infection. Horizontal lines within data points depict mean values. *<i>P</i> = 0.036, Mann-Whitney test.</p

Schematic of BLT humanized mouse generation and timeline of HIV-1 infection and anti-PD-1 mAb treatment.

<p>Schematic of BLT humanized mouse generation and timeline of HIV-1 infection and anti-PD-1 mAb treatment.</p

PD-1 expression on T cells in HIV-infected BLT mice.

<p>Peripheral blood was obtained at various time points after HIV infection. <b>A</b>) Percentages of human CD8⁺ and CD4⁺ T cells expressing PD-1. Horizontal lines within data points depict mean value. Uninfected controls were from peripheral blood samples obtained at time points when littermates were infected for 9-13 weeks. (HIV infected: n = 9 mice at wk 3, n = 18 mice at other time points; Uninfected: n = 7 mice). *<i>P</i> = 0.01, Wilcoxon matched pairs test; **<i>P</i> = 0.001, Mann-Whitney test. <b>B</b>) Representative flow cytometry data of PD1 expression on CD8⁺ or CD4⁺ T cells at 13 weeks post infection. PD1-HI representative is mouse #4 and PD1-LO is mouse #1 depicted in the next panel. <b>C</b>) Percentages of CD8+ and CD4+ T cells expressing PD-1 in PD1-LO (defined as having <30% PD-1⁺CD8⁺ cells) and PD1-HI (>30% PD-1⁺CD8⁺ cells).</p

Effects of anti-PD-1 mAb or control treatment on HIV viral loads in chronically infected BLT mice.

<p>BLT mice infected with HIV-1 for 13 weeks were injected intraperitoneally with anti-PD-1 mAb, control mAb, or no Ab on days 0, 3, 7 and 10 (200 µg/dose, arrow). Peripheral blood was collected at multiple time points and HIV-1 plasma viral load was measured by quantitative RT-PCR. Graph represents mean viral load of Control (n = 10, control mAb or no Ab), anti-PD-1 mAb-treated PD1-LO mice (n = 3), and anti-PD-1 mAb-treated PD1-HI mice (n = 5). *<i>P</i><0.05, Mann-Whitney test.</p

HIV-1 induces HBD1 <i>in vitro</i> but is unlikely to be sufficient <i>in vivo</i>.

<p>(A-C) CD14+ monocytes isolated using Miltenyi MACS technology were incubated with either HIV-1 R5, poly I:C (TLR3 ligand), 5’ppp-dsRNA (RIG-I ligand), R837 (TLR7 ligand), LPS (TLR4 ligand) or FLA-ST (TLR5 ligand), or left untreated. After 24 h cells were harvested and RNA extracted. Relative expression of HBD1 (A, C) or HBD2 (B) was assessed using quantitative PCR. HBD1 expression was found to be significantly increased in monocytes when incubated with HIV-1 R5, poly I:C or 5’pppdsRNA (A), whereas LPS and FLA-ST induced HBD2 (B). (C) Significant upregulation of HBD1 by HIV-1 was only achieved at 1x10^6 infection HIV-1 particles (i.p.) (Kruskal-Wallis and Dunn’s multiple post comparison test). Data points are presented with median and interquartile range which each dot representing an independent experiment from a different healthy control subject. (D) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.g001" target="_blank">Fig 1A</a>) was plotted against the corresponding viral load which each dot representing one subject. No significant correlation was found between viral load and HBD1 transcription (Spearman r correlation). (E, F) CD4+ T cells or CD14+ monocytes were isolated from PBMCs and incubated with either β-lactamase packaged HIV-1 X4 (CXCR4 tropic)/ HIV-1 R5 (CCR5 tropic) or left untreated. Where indicated cells were pre-treated with the CCR5 antagonist maraviroc (Mav) for 30min before HIV-1 treatment. All virus contained Vpr-BlaM which allowed assessment of HIV-1 entry into cells 12h after exposure. The highest entry was observed in CD4+ T cells by HIV-1 X4, whereas HIV-1 R5 entered CD4+ T cells as well CD14+ monocytes at low frequency. The graph shows the median and interquartile range with each dot representing one independent experiment from a different healthy control subject (F).</p

IFN-α is a potent inducer of HBD1 in monocytes <i>in vitro</i> and correlates with HBD1 transcription <i>in vivo</i>.

<p>(A, B) CD14+ monocytes (A n = 11 and B n = 5) were isolated from PBMCs and incubated with different concentrations of recombinant IFN-α2 or left untreated. Where indicated cells were treated with either an anti-IFN-α or an isotype control antibody 30 min before stimulation with 50 pg/ml recombinant IFN-α2. Relative expression of HBD1 and ISG15 was assessed using quantitative PCR. IFN-α2 was found to significantly upregulate HBD1 and ISG15. Each dot represents one independent experiment from a different healthy control subject. (C, D) Human PBMCs were isolated from whole blood from HIV-1 uninfected (HIV-) (C n = 5, D n = 9), HIV-1 untreated chronic progressors (PG) (C n = 9, D n = 14) and acutely HIV-1 infected (Acute) individuals (C n = 12, D n = 15). IFNα (IFNA) transcription of PBMCs was assessed by qPCR. IFNA and ISG15 were both significantly upregulated in acutely but not chronically infected subjects compared to HIV-uninfected control subjects (A-D) Kruskal-Wallis and Dunn’s multiple comparison test with graphs showing median and interquartile range. (E, F) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.g001" target="_blank">Fig 1A</a>) was plotted against the corresponding transcription of IFNA (E) or ISG15 (F) with each dot representing one subject. ISG15 and IFNA were found to significantly correlate with HBD1 transcription in acutely infected individuals (Spearman r correlation).</p

HBD1 is expressed by circulating monocytes and gut epithelial cells.

<p>(A, B) Cells were sorted from 5 different bulk colon excess tissue (n = 5) or PBMCs (n = 5), RNA extracted and HBD1 transcription quantified by qPCR. For colon, epithelial cells were identified using the surface marker CD326 and CD45 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). For PBMCs, monocytes were identified as CD14+, DCs as CD11c+ (mDCs) or CD123+ (pDCs) and lymphocytes as CD3+CD19+ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). Epithelial cells were the main producers of HBD1 in colon whereas CD14+ cells were the main producers in PBMCs. (C, D) The frequency of HBD1 producing cells is not altered during acute infection. Frequency of monocytes (C) and DC (D) populations in PMBCs from HIV-1- (n = 9) or acutely (Acute; n = 8) infected individuals was determined by flow cytometry. Conventional monocytes were identified as CD14+ CD16-, inflammatory monocytes as CD14+ CD16+ and mDCs as well as pDCs as described above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). No significant difference in cell frequencies was found between HIV-1- and sample from subjects with acute HIV-1 infection for any of the analyzed cell populations (Kruskal-Wallis and Dunn’s multiple post comparison test). (A-D) Data points are presented with median and interquartile range.</p

HBD1 is upregulated in PBMCs during acute HIV-1 infection.

<p>Samples from HIV-1 uninfected (HIV-), HIV-1 untreated chronic progressors (PG), HIV-1 chronic ART treated (CT) and acutely HIV-1 infected (Acute) individuals were analyzed for HBD1 expression. HBD1 transcription in PBMCs (A, B) or gut pinch biopsies (C, D) was determined by qPCR. Grey color indicates subjects with matched gut biopsy samples. (A) HBD1 expression in blood of acutely infected subjects (n = 32) was found to be significantly higher compared to HIV-1 individuals from PG (n = 8), CT (n = 9) or HIV- (n = 17) subjects (Kruskal-Wallis and Dunn’s multiple comparison test with error bars indicate min and max of boxplots). (B) Longitudinal sampling of individuals (n = 10) showed significant downregulation of HBD1 in chronic HIV-1 infection compared to acute infection (Wilcoxon matched-pairs signed rank test). (C, D) No significant differences in HBD1 expression in ileum (C) or colon (D) was found between HIV- (n = 5), PG (n = 4), CT (n = 9) and HIV acute infected subjects (n = 4), (Kruskal-Wallis and Dunn’s multiple comparison test and median with interquartile range).</p

NQS filtering improves fit of probability model to data.

<p>(<b>A</b>) Quantile-quantile (q-q) plots under NQS filtering show good fit of the probability model to the observed distribution of errors. Since the probability model is discrete, p values are projected onto a uniform distribution, and the distribution of projected p values is compared with the expected null distribution. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002417#s4" target="_blank">Materials and Methods</a> section for details. (<b>B</b>) In contrast, q-q plots under no filtering show that no filtering skews the calibration of the probability model used by <i>V-Phaser</i>. Q-q plots of models based on subsets of the reads demonstrate that this effect becomes more pronounced with increasing coverage (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002417#pcbi.1002417.s001" target="_blank">Figure S1</a>). Q-q plots are scaled to fit curve, so y = x line is not at a 45 degree angle.</p

Phase information increased sensitivity, and base quality scores increased specificity.

<p>We compared <i>V-Phaser</i> to alternate versions of <i>V-Phaser</i> with specific components disabled. In the No Phase version, <i>V-Phaser</i> called variants without phase information. In the Uniform Errors version, <i>V-Phaser</i> estimated uniform error rates within homopolymer and nonhomopolymer regions without regard to assigned base qualities. In the No Filtering version, <i>V-Phaser</i> did not filter out low quality bases. (<b>A</b>) Phase information increased sensitivity. The version without phase information attained a sensitivity of 90%, but all other versions of <i>V-Phaser</i> used phase information and attained a sensitivity of 97% or more. We calculated sensitivity as the percentage of known variants correctly identified. Data are from WNV mixed population control dataset. (<b>B</b>) Individual base quality scores increased specificity. Among loci with mismatches, the Uniform Errors version had only 91% specificity, but all other versions incorporated base quality scores in their probability model and attained 97% specificity or more. We calculated specificity as the percentage of loci in the control sample correctly identified as having no variants among loci that had at least one candidate variant. Data are from infectious clone (HIV NL4-3) control dataset.</p